Antibody therapeutics are frequently designed by optimising an initial lead antibody in order to select desired characteristics, such as binding affinity, Kd, or lack of immunogenicity. Frequently human antibodies are generated in animals such as transgenic mice expressing human immunoglobulin genes.
After generation and isolation of a lead candidate antibody, the antibody may be optimized in various ways. Typically the lead antibody is sequenced, and the sequence used to generate an antibody library of variants for further screening. The variants may be constructed using oligonucleotides to introduce degeneracy into the coding regions (for example, the regions coding for one or more of the CDRs). The oligonucleotides may be used for PCR amplification of regions of the nucleic acids coding for the antibody. This will typically generate a large library including many variants, in which each amino acid residue in the lead is replaced with many potential substitutions. The libraries may then be cloned into expression vectors in order to generate the antibodies themselves, Display systems such as ribosome, phage or yeast display systems may be used. The antibodies thereby produced can then be screened for improvements in the desired properties.
A drawback with these known methods is that potentially far more variants are generated than will show desirable properties. This increases the time and resources necessary to generate the library and to select a variant antibody with desired characteristics. Furthermore, optimising both heavy and light chains of a fully human antibody increases the necessary workload, as well as introducing further uncertainty as to the properties of the antibody, particularly for antibodies that have both heavy and light immunoglobulin chains when assembled.
The present invention is intended to address at least some of these disadvantages, and to provide an additional method for generating immunoglobulin libraries. This is achieved in part through the effective pre-selection of certain variants by the immunised animal itself by the process of somatic hypermutation. During generation of native antibodies, proliferation of B cells is accompanied by an extremely high rate of somatic mutation in the B cell receptor locus, which generates the required antibody diversity. The mutations are mainly concentrated at certain somatic hypermutation hotspots. The present invention makes use of this native generation of diversity in order to inform the design of the immunoglobulin library.